High bacterial density and diversity near plant roots has been attributed to rhizodeposit compounds that serve as both energy sources and signal molecules. However, it is unclear if and how specific rhizodeposit compounds influence bacterial diversity. We silenced the biosynthesis of isoflavonoids, a major component of soybean rhizodeposits, using RNA interference in hairy-root composite plants, and examined changes in rhizosphere bacteriome diversity. We used successive sonication to isolate soil fractions from different rhizosphere zones at two different time points and analyzed denaturing gradient gel electrophoresis profiles of 16S ribosomal RNA gene amplicons. Extensive diversity analysis of the resulting spatio temporal profiles of soybean bacterial communities indicated that, indeed, isoflavonoids significantly influenced soybean rhizosphere bacterial diversity. Our results also suggested a temporal gradient effect of rhizodeposit isoflavonoids on the rhizosphere. However, the hairy-root transformation process itself significantly altered rhizosphere bacterial diversity, necessitating appropriate additional controls. Gene silencing in hairy-root composite plants combined with successive sonication is a useful tool to determine the spatio temporal effect of specific rhizodeposit compounds on rhizosphere microbial communities.Pioneering microbiology studies by L. Hiltner in the early 1900s showed that the highest microbial density in soils occurs very close to plant roots (Hinsinger and Marschner 2006). For example, a four-to fivefold increase in CFU was observed in root-surface scrapings as compared with soil samples 0.5 cm away from the roots (Clark 1940). Such changes are attributed to the rich carbon energy sources provided by the plant. Indeed, plants release, on average, 10 to 15% (Jones et al. 2009) of their photosynthetic assimilates into the rhizosphere, a process called rhizodeposition (Dennis et al. 2010). These rhizodeposits originate from sloughed off root border and root border-like cells from root tips, active root exudation, and cell lysis. Rhizodeposits are composed of sugars, amino acids, organic acids, fatty acids, proteins, ions, secondary metabolites, mucilage, water, and miscellaneous carbon-containing compounds (Bais et al. 2006;Dennis et al. 2010).Significant evidence accumulated over the years indicates that the composition of root microbial communities is influenced, in large part, by the plant species and its developmental stage (Micallef et al. 2009;Mougel et al. 2006;Weisskopf et al. 2006). Indeed, an intricate coevolution of plants and rhizosphere microbial communities was suggested by the observation that resident plants or their root exudates are capable of maintaining the biomass and diversity of soil fungal communities to a much greater extent than nonresident or introduced plants ). This is supported by the observation that invasive weeds have the ability to significantly influence native rhizosphere microbial communities to exert their dominance in new environments (Inder...